It is now recognized that cyclic GMP (cGMP) is an important regulator of mammalian vascular smooth muscle function. Physiologically important regulators of smooth muscle tone such as the endothelium-derived relaxing factor and atrial natriuretic factor relax vascular smooth muscle by increasing the cellular levels of cGMP. The mechanism by which cGMP relaxes vascular smooth muscle is still incompletely understood, but one clear effect of the nucleotide is to reduce intracellular levels of calcium. This results in the dephosphorylation of myosin light chains and a return of the cell to the uncontracted state. The cGMP receptor protein which mediates the reduction of calcium is the cGMP-dependent protein kinase (cG kinase), but the mechanism by which the enzyme causes reduction in calcium is unknown. In order to elucidate the role of cG kinase in smooth muscle relaxation, the effects of this enzyme on protein phosphorylation and calcium levels will be examined in cultured vascular smooth muscle cells. The identifies of phosphoproteins will be ascertained in broken cell systems using amino acid sequence information and the effects of these substrate proteins on membrane calcium pumps. In addition, the effects of cG kinase on membrane phospholipid levels will be examined in the intact cell in order to study the relationship between calcium pump activity and lipid phosphorylation. To further understand the actions of cG kinase, the cDNA for the enzyme will be cloned, sequenced, and ligated into expression vectors. These vectors will be used to (i) overproduce cG kinase in pro- and eukaryotic cells to obtain ample material for further study, and (ii) transfect cultured muscle cells and examine biochemical and phenotypic changes which result from the transfection. Finally, the regulation of cG kinase expression in cultured aortic smooth muscle cells will be studied using cDNA probes. The effects of contractile agonists, growth and differentiation factors, mechanical stretch, and endothelial cells on the cG kinase and its mRNA levels will be examined in cells. These studies will provide a basis for understanding the molecular actions of cG kinase and factors which regulate its expression in vascular smooth muscle. The knowledge gained for this project will advance the current understanding of normal smooth muscle function. Because vascular diseases such as hypertension and atherosclerosis are characterized in part at least by decreased cGMP production in smooth muscle cells, the proposed studies may lead to new approaches toward the treatment of vascular diseases.